cnm-positive Streptococcus mutans is associated with galactose-deficient IgA in patients with IgA nephropathy

The presence of Streptococcus mutans expressing Cnm protein encoded by cnm (cnm-positive S. mutans) in the oral cavity is associated with immunoglobulin A (IgA) nephropathy (IgAN). However, the precise mechanism by which cnm-positive S. mutans is involved in the pathogenesis of IgAN remains unclear. The present study evaluated glomerular galactose-deficient IgA1 (Gd-IgA1) to clarify the association between the presence of cnm-positive S. mutans and glomerular Gd-IgA1 in patients with IgAN. The presence of S. mutans and cnm-positive S. mutans was evaluated by polymerase chain reaction in saliva specimens from 74 patients with IgAN or IgA vasculitis. Immunofluorescent staining of IgA and Gd-IgA1 using KM55 antibody in clinical glomerular tissues was then performed. There was no significant association between the glomerular staining intensity of IgA and the positive rate of S. mutans. However, there was a significant association between the glomerular staining intensity of IgA and the positive rate of cnm-positive S. mutans (P < 0.05). There was also a significant association between the glomerular staining intensity of Gd-IgA1 (KM55) and the positive rate of cnm-positive S. mutans (P < 0.05). The glomerular staining intensity of Gd-IgA1 (KM55) was not associated with the positive rate of S. mutans. These results suggest that cnm-positive S. mutans in the oral cavity is associated with the pathogenesis of Gd-IgA1 in patients with IgAN.

Although the precise mechanism is still unknown [5], several studies have suggested that galactose-deficient IgA1 (Gd-IgA1) is a key effector molecule in the pathogenesis of IgAN [6][7][8][9]. A novel lectin-independent enzyme-linked immunosorbent assay (ELISA) using an anti-Gd-IgA1 monoclonal antibody (KM55) was recently developed [10]. Glomerular Gd-IgA1 deposition has been shown by immunofluorescence with KM55 antibody, which provides new insights into the possibility that Gd-IgA1 functions as a key effector molecule of IgAN [10,11]. Gd-IgA1 has been specifically detected in IgAN and IgA vasculitis, but not in other renal diseases [11].
Whether cnm-positive S. mutans is related to the production of Gd-IgA1 when IgAN is induced is unknown. If cnm-positive S. mutans proves to be associated with Gd-IgA1, this is a major discovery and could be a breakthrough in determining the pathogenic mechanism of IgAN. Although an association between bacterial infection and IgAN is assumed [13][14][15][16][17], there is no evidence that Gd-IgA1 production is involved in the mechanism of IgAN induced by infection.
In the present study, we analyzed Gd-IgA1 using KM55 antibody to clarify the association between the presence of cnm-positive S. mutans in the oral cavity and glomerular Gd-IgA1.

Patients and clinical characteristics
Seventy-eight patients who underwent a renal biopsy at Seirei Hamamatsu General Hospital, Hamamatsu, Japan in 2017-2021 were initially included. All patients were over 18 years of age. These patients were diagnosed with IgAN (n = 68) or IgA vasculitis (Henoch-Schonlein purpura nephritis) (n = 10) by renal biopsies. Their diagnoses were made on the basis of light microscopy and immunohistochemical findings. Four of the 78 patients were excluded because there were no glomeruli in prepared sections for Gd-IgA1 (KM55) staining. Therefore, analysis was performed on 74 patients. Clinical data (age, sex, height, body weight, body mass index, blood pressure, serum creatinine, estimated glomerular filtration rate [eGFR], serum C3, serum IgA, urinary protein [g/g creatinine], and urinary sediment of red blood cells [RBCs] [>100/high-power field]) were obtained at the time of renal biopsy after patients had provided informed consent to participate in this study.

Analysis of cnm-positive S. mutans
Saliva specimens obtained from the patients and frozen at -20˚C were used to test for the presence of S. mutans DNA and whether it was cnm-positive or cnm-negative by polymerase chain reaction, as described previously [21]. Campylobacter rectus and Porphyromonas gingivalis DNA was also evaluated, as described elsewhere [38].

Measurement of Gd-IgA1
Plasma was collected at the time of the renal biopsy. Plasma Gd-IgA1 concentrations were evaluated using the KM55 ELISA kit (Immuno-Biological Laboratories Co., Ltd). Briefly, the ELISA plates were incubated with plasma specimens (1:200 dilution in enzyme immunoassay buffer) and standard specimens for 1 hour at room temperature, washed 4 times with wash buffer, incubated with prepared-labeled antibodies, and then treated with 50 ml of tetramethylbenzidine solution for 30 minutes in the dark. Absorbance was evaluated at 450/650 nm by the Versamax Microplate Reader (Molecular Devices, Tokyo, Japan). The Gd-IgA1 concentrations was evaluated according to the standard curve.

Statistical analysis
All the results are expressed as the mean ± standard deviation (SD). When there was a significant difference, a further statistical analysis was conducted using Fisher's PLSD test or Fisher's exact test between 2 groups. The Cochran-Armitage trend test was used for trend analysis. A simple regression analysis was used for correlation analysis. In these analyses, P < 0.05 was considered statistically significant. The statistical analyses were conducted using Statview software (SAS Institute Inc., Cary, NC, USA) and GraphPad Prism 8 software (San Diego, CA, USA).

Glomerular staining intensity of IgA and Gd-IgA1
The glomerular staining intensity of IgA and Gd-IgA1 was defined by the intensity of 0 to +3 as shown in Fig 1. IgA and Gd-IgA1 were mainly positive in the mesangial region, and IgA and Gd-IgA1 had the same distribution. There was a significant association between the glomerular staining intensity of IgA and Gd-IgA1 using regression analysis (R = 0.787, P < 0.001).
Plasma Gd-IgA1 concentrations were measured in 33 of 74 patients. There was no significant association between the glomerular staining intensity of Gd-IgA1 and plasma Gd-IgA1 concentrations (n = 33) (S1 Fig).

Association between glomerular staining intensity of IgA and Gd-IgA1, and the rate of cnm-positive S. mutans in the oral cavity
There was no significant association between the glomerular staining intensity of IgA and the positive rate of S. mutans (Fig 2A). However, there was a significant association between the glomerular staining intensity of IgA and the positive rate of cnm-positive S. mutans (Fig 2B). There was also no significant association between the glomerular staining intensity of Gd-IgA1 and positive rate of S. mutans (Fig 2C). However, there was a significant association between the glomerular staining intensity of Gd-IgA1 and the positive rate of cnm-positive S. mutans (Fig 2D).
There was no significant association between the glomerular staining intensity of IgA and the positive rate of C. rectus (S2A Fig)

Comparison of the cnm-positive S. mutans and cnm-negative S. mutans groups
The percentage of the staining intensity of IgA: 3+ was significantly higher in the cnm-positive S. mutans group than in the cnm-negative S. mutans group ( Table 1). The percentage of the staining intensity of Gd-IgA1: 3+ was also significantly higher in the cnm-positive S. mutans group than in the cnm-negative S. mutans group (Table 1). No significant differences were found in age, sex, height, body weight, body mass index, systolic blood pressure, diastolic blood pressure, serum creatinine concentrations, eGFR, serum C3 concentrations, serum IgA concentrations, or urinary protein concentrations between the groups ( Table 1). The percentage of urinary sediment �100 RBCs/high-power field was higher in the cnm-positive S. mutans group than in the cnm-negative S. mutans group, but this was not significant (Table 1). No significant differences were found in the mesangial hypercellularity score, endocapillary hypercellularity score, segmental glomerulosclerosis score, tubular atrophy/interstitial fibrosis score, or the crescent score of the Oxford classification between the groups ( Table 2).

Discussion
As far as we know, this is the first study to demonstrate an association between cnm-positive S. mutans in the oral cavity and glomerular Gd-IgA1. We discovered that there was a significant association between the positive rate of cnm-positive S. mutans in the oral cavity and glomerular staining intensity of IgA or Gd-IgA1. Although various factors are considered to be , and 3+ staining intensity. Seventy-four patients were divided into four groups according to the staining intensity of IgA: group 0 (n = 4), group 1+ (n = 46), group 2+ (n = 12), and group 3+ (n = 12). These patients were also divided into four groups according to the staining intensity of Gd-IgA1: group 0 (n = 4), group 1+ (n = 35), group 2+ (n = 23), and group 3+ (n = 12). Fluorescein-conjugated polyclonal rabbit anti-human IgA antibody (DAKO Japan Inc., Kyoto, Japan), anti-human Gd-IgA1 antibody (KM55) (Immuno-Biological Laboratories Co., Ltd, Gunma, Japan), and Alexa Fluor 555-conjugated goat anti-rat IgG antibody (Life Technologies) was used for immunofluorescence staining. Original magnification: ×200. Gd-IgA1, galactose-deficient IgA1.
https://doi.org/10.1371/journal.pone.0282367.g001 associated with Gd-IgA1, these data indicate that cnm-positive S. mutans in the oral cavity may be one of the factors associated with the development of IgA nephropathy via Gd-IgA1.
Recent clinical studies have demonstrated that cnm-positive S. mutans in the oral cavity is associated with IgAN [20][21][22]26]. One study showed that the positive rate of cnm-positive S. mutans in the oral cavity was significantly higher in the IgAN group than in the healthy control group (32.1% vs. 14.0%) [20]. However, the positive rate of S. mutans was similar between the IgAN and healthy control groups [20]. Another study suggested that cnm-positive S. mutans in the oral cavity and the dental caries status were associated with urinary protein in patients with IgAN [21]. The Cnm protein in the tonsils may be associated with the severity of IgAN [22]. The report demonstrated the following: (1) the positive Cnm protein area/total tonsillar area rate was significantly higher in patients with IgAN than in the control (chronic tonsillitis) group; (2) Cnm protein existed in the tonsils, not in the glomerulus, in patients with IgAN; and (3) Cnm protein in the tonsils was associated with urinary protein in patients with IgAN [22]. These results suggest that IgAN is aggravated by immune reactions in the tonsils via an unknown mechanism induced by Cnm protein [22]. In rodent models, intravenous  [23] and severe dental caries induced by cnm-positive S. mutans caused IgAN-like glomerulonephritis [24]. As mentioned above, we found an association between cnm-positive S. mutans and IgAN. However, we could not identify how cnm-positive S. mutans induced IgAN.
Gd-IgA1 has been demonstrated as a key effector molecule in the pathogenesis of IgAN [6][7][8][9]. We showed an association between the presence of cnm-positive S. mutans and glomerular Gd-IgA1 in this study. This study indicates that cnm-positive S. mutans induced Gd-IgA1 production in the process of causing IgAN, suggesting that the most upstream factor for Gd-IgA1 production is oral bacterial infection, such as cnm-positive S. mutans. We hypothesize that repeated immune reactions involving IgA induced by infection such as cnm-positive S. mutans in the mucosal immune tissues of the oral cavity (e.g., the tonsils) may cause Gd-IgA1 [21]. We believe that this result is important in considering the pathogenic mechanism of IgAN. We do not have enough evidence to explain how cnm-positive S. mutans affects the induction of Gd-IgA1 at this stage and need to elucidate these mechanisms in a further study.
We also investigated the positive rate of C. rectus and P. gingivalis in the oral cavity because these bacteria are reported to be potentially associated with IgA nephropathy [19,26,27]. We could not confirm the association between these bacteria and Gd-IgA1 in this research. Our study showed that the percentage of urinary sediment �100 RBCs/high-power field was higher in the cnm-positive S. mutans group than in the cnm-negative S. mutans group. Although this finding did not reach statistical significance, it suggests that cnm-positive S. mutans is associated with the hematuria that is the cardinal symptom of IgAN.
Unfortunately, because the exact mechanism of IgAN is not yet understood, there are no specific disease-targeted therapies for IgAN [5]. Our present findings are consistent with the new concept of an oral-kidney association and could establish a new treatment for IgAN [21]. Considering that S. mutans is a pathogen that causes dental caries, reducing it in the oral cavity to prevent dental caries may improve the prognosis of IgAN [27]. Further studies are needed to elucidate the effects of oral care as a treatment of IgAN.
There are some limitations in this study. First, although we demonstrated that cnm-positive S. mutans in the oral cavity was associated with Gd-IgA1 in patients with IgAN, how cnm-positive S. mutans contributes to Gd-IgA1 needs to be elucidated. Second, given that the only S. mutans protein we examined was Cnm, the possibility that other S. mutans proteins are associated with Gd-IgA1 cannot be excluded. Third, the sample size was small, and all patients were of the same ethnicity and from a single facility. Further studies in larger numbers of ethnically diverse patients from multiple facilities are needed to confirm our present findings.
In conclusion, cnm-positive S. mutans in the oral cavity is associated with Gd-IgA1 in glomeruli in patients with IgAN. This finding suggests that cnm-positive S. mutans is involved in the pathogenicity of IgAN through induction of Gd-IgA1. no. 1704-036). All patients were informed of the study protocol, and provided written